Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose

Abstract

This invention provides novel enzyme compositions using newly identified and isolated C. lucknowense enzymes, including CBH Ib CBH IIb, EG II, EG VI, β-glucosidase, and xylanase II in conjunction with previously identified enzymes CBH Ia, CBH IIa (previously described as Endo 43), and EG V. These enzyme compositions demonstrate an extremely high ability to convert lignocellulosic biomass (e.g., Avicel, cotton, Douglas fir wood pretreated by organosolv) to glucose. CBH Ia and IIb, which both have a cellulose-binding module (CBM) displayed a pronounced synergism with three major endoglucanases (EG II, EG V, EG VI) from the same fungus in hydrolysis of cotton as well as a strong synergy with each other. The enzyme compositions are effective in hydrolysis of the lignocellulosic biomass.

We claim: 
     
       1. A mutant  Chrysosporium lucknowense  strain capable of expressing at least one cellobiohydrolase and at least one endo-1,4-β-glucanase at higher levels than the corresponding non-mutant strain under the same conditions;
 wherein at least one cellobiohydrolase is selected from the group consisting of CBH Ia, CBH Ib, CBH IIa and CBH IIb; and wherein at least one endo-1,4-β-glucanase is selected from the group consisting of EG II, EG V, and EG VI. 
 
     
     
       2. The mutant  Chrysosporium lucknowense  strain according to  claim 1 , wherein said strain is capable of expressing a β-glucosidase and/or a xylanase at higher levels than the corresponding non-mutant strain under the same conditions. 
     
     
       3. The mutant  Chrysosporium lucknowense  strain according to  claim 2 , wherein said xylanase is Xyl II. 
     
     
       4. The mutant  Chrysosporium lucknowense  strain according to  claim 1 , wherein said mutant is or is derived from a  Chrysosporium lucknowense  mutant strain selected from the group consisting of  Chrysosporium lucknowense  strain C1 (Deposited as Accession Number VKM F-3500 D), UV13-6 (Deposited as Accession Number VKM F-3632 D), NG7C-19 (Deposited as Accession Number VKM F-3633 D), and UV18-25 (Deposited as Accession Number VKM F-3631 D). 
     
     
       5. The mutant  Chrysosporium lucknowense  strain according to  claim 1 , wherein at least one of said EG II, EG V, and EG VI one endo-1,4β-glucanases are selected from the group consisting of EG II (SEQ ID NO: 10), EG V (SEQ ID NO: 14), EG VI (SEQ ID NO: 16). 
     
     
       6. The mutant  Chrysosporium lucknowense  strain according to  claim 1 , wherein at least one of said CBH Ia, CBH Ib, CBH IIa and CBH IIb are selected from the group consisting of CBH Ib (SEQ ID NO: 2), CBH IIb (SEQ ID NO: 4), EG II (SEQ ID NO: 10), EG V (SEQ ID NO: 14), EG VI (SEQ ID NO: 16), BGL (SEQ ID NO: 12), and Xyl II (SEQ ID NO: 18). 
     
     
       7. The mutant  Chrysosporium lucknowense  according to  claim 1 , wherein at least one of said cellobiohydrolase and at least one endo-1,4-β-glucanase have at least 65% amino acid identity as determined by the BLAST algorithm with at least one of the following:
 EG VI amino acid sequence of SEQ ID NO: 16 or a part thereof having at least 20 contiguous amino acids; 
 the BGL amino acid sequence of SEQ ID NO: 12 or a part thereof having at least 20 contiguous amino acids; 
 the Xyl II amino acid sequence of SEQ ID NO: 18 or a part thereof having at least 20 contiguous amino acids. 
 
     
     
       8. A modified microorganism or plant capable of expressing one or more of an enzyme selected from the group consisting of CBH Ib (SEQ ID NO: 2), CBH IIb (SEQ ID NO: 4), EG II (SEQ ID NO: 10), EG V (SEQ ID NO: 14), EG VI (SEQ ID NO: 16), BGL (SEQ ID NO: 12), and Xyl II (SEQ ID NO: 18) and the second enzyme is a hemicellulase, cellobiohydrolase, endo-1,4-βglucanase, β-glucosidase or a xylanase. 
     
     
       9. The modified microorganism according to  claim 8 , wherein said microorganism is a fungus. 
     
     
       10. The modified microorganism according to  claim 9 , wherein said fungus is selected from the group consisting of  Talaromyces, Aspergillus, Trichoderma, Neurospora, Penicillium, Fusarium, Humicola, Myceliophthora, Corynascus, Chaetomium, Tolypocladium, Thielavia, Acremonium, Sporotrichum, Thermoascus, and Chrysosporium.    
     
     
       11. The modified microorganism according to  claim 10 , wherein said fungus of the genus  Chrysosporium  is a strain selected from the group consisting of the wild-type  Chrysosporium  strain C1 , Deposited as Accession Number VKM F-3500 D; mutant C1 strain UV13-6, Deposited as Accession Number VKM F-3632 D; mutant C1 strain NG7C-19, Deposited as Accession Number VKM F-3633 D; and mutant C1 strain UV18-25, Deposited as Accession Number VKM F-3631 D.